Note: Descriptions are shown in the official language in which they were submitted.
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1
AVIAN SEX DETERMINATION METHOD
The present invention relates to a method for sexing individual subjects of
avian
species.
Adults and particularly offspring of many avian species are monomorphic,
making
determination of sex difficult. Nucleic acid probes that hybridise to the DNA
of the
female-specific W chromosome have lead to molecular solutions to this problem
for
some species. However, use of such techniques has proved to be difficult and
in many
cases, their taxonomic range is limited. More recently, polymerase chain
reaction
(PCR) based approaches that are technically simpler and that have broader
taxonomic
utility have been developed.
Sex identification methods have also been based upon examining differences in
intron
size between the female W specific chromosome and the Z chromosome, which
occurs
in both sexes (female, ZW; male, Z~ (Ellegren 1996, Kahn et al 1998). Another
approach has been to identify specific genes located on the W chromosome.
Analysis
of the chromobox-helicase-DNA-binding gene (CIiD) shows that it contains
sequences specific to the W chromosome and can be used for determination of
the sex
of most birds (Griffiths, et al 1998). A combination of the analysis of intron
size
difference between sexes and the chromosome specific CHD gene has also been
proposed (Fridolfsson, A-K and Ellegren, H, 1999). Another method proposed has
been to analyse W-specific repeat sequences in order to determine the sex of
chick
embryos (Clinton, 1994). However, this method required separate sexing and
control
PCR reactions. A rapid and simple single tube chicken sexing protocol based on
a
PCR analysis of W chromosome specific sequences has been devised more recently
(Clinton et al 2001). Other methods have been proposed in WO 96/39505 based on
an
analysis of DNA sequences (introns and exons) encoding two genes located on
the Z
and W chromosomes of birds. Other means for analysis have been proposed in US-
A-
5,679,514 and US-A-5,707,809.
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2
Such methods of sex determination generally require technical expertise and
specialist
facilities, and are not susceptible to ready automation, especially in
agricultural
environments. The sequences referred to also have variants (homologues in the
case
of genes) of that sequence present on the Z-chromosome (i.e. the avian sex
chromosome that is found in both sexes).
The avian gene WPKCI has been shown to be conserved widely on the avian W
chromosome and expressed actively in the female chicken embryo before the
onset of
gonadal differentiation. It is suggested that WPKCI may play a role in the
differentiation of the female gonad by interfering with the function of PKCI
or by
exhibiting its unique function in the nucleus (Hori et al 2000). This gene has
also
been identified as ASW (avian sex-specific W-linked) (O'Neill et al 2000).
Accurate determination of the sex of avians is a particularly important issue
for the
poultry industry for both economic and welfare reasons. Companies which
produce
egg-layer strains of chickens ("layers") would prefer to be able to
(inexpensively)
determine the sex of birds at hatch and just raise the females; companies
which
produce meat strains of chickens ("broilers") would prefer to (inexpensively)
sex birds
at hatch and just raise males as they grow much faster and eat less.
Currently, most
"broiler" producers accept the inefficiency of producing and rearing female
birds,
whilst a proportion of the "layer" producers use relatively expensive
procedures to
determine the sex of one-day old chicks.
The use of sex determination procedures on one-day old chicks has significant
welfare
implications, in particular the disposal of unwanted male chicks from layer
strains of
chickens. Recent estimates suggest that at least 280 million such chicks have
to be
disposed of each year in the European Union alone. The means of disposal used
in
practice are killing the chicks by maceration, or by gassing or by
electrocution (both of
the latter followed by incineration). The use of maceration has been
recommended as
the other can techniques leave approximately 40% of the chicks alive prior to
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3
mcmeration.
There exists a need, therefore, for simple, accurate methods that can be used
at poultry
farms that overcome the problems in the prior art and allow for improved
animal
welfare.
It has now been surprisingly discovered that a W-chromosome specific
transcript can
be used as the basis for a sex determination method that overcomes the
problems
previously encountered in this field to date. The W-specific transcript is
surprising as
it is 3' to 5' in relation to the transcribed strand (5' to 3') for the gene
WPKCI already
known and there is no known Z-chromosome copy.
According to a first aspect of the invention, there is provided a method for
the
determination of the sex of an avian subject, the method comprising contacting
a
sample from said subject with a nucleic acid probe comprising an at least 6
base pair
fragment from a target nucleic acid sequence as shown in Figures 8 to 14, or a
sequence complementary or homologous thereto.
The present invention provides methods for the determination of the sex of an
avian
subject, i.e. whether the subject is male or female. The methods of the
present
invention can be used to determine the sex of a subject of the Class Aves, for
example
bird species of agricultural importance such as Gallus gallus (chiclcen),
turkeys, quail,
guinea fowl, commonly referred to as poultry. Such methods may find
application in
relation to other bird species, such as those bred in captivity, kept as
domestic pets, or
kept in zoological institutions and examples include penguins, parrots, and
rare bird
species threatened with extinction, and/or the subjects of breeding programmes
for
conservation. The subject avian being analysed may be an embryo, a newly
hatched
chick or a mature adult bird.
Samples that can be assayed according to a method of the present invention
include
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4
but are not limited to samples of allantois or amnion from the egg, i.e.
allantoic fluid
or amniotic fluid, of an avian subject containing a developing embryo; other
sources of
suitable samples include any convenient sample of a biological nature
containing cells,
tissue or organs, for example, muscle, heart, brain, lung, liver,
chorioallantoic
membrane, mesonephrous and blood.
Such methods can be carried out on samples removed from an egg without
compromising the viability of the egg using standard procedures. Samples may
be
removed from the egg manually or by using an automated approach. Machines
intended for delivery of vaccine to eggs for incubation can be altered to
sample the
fluids of the egg in the same manner. The methods can, of course, be performed
equally on cultured cells in vitro.
The samples to be analysed according to a method of the present invention, may
be
analysed by means of a DNA amplification procedure, such as the polymerase
chain
reaction (PCR), or by means of RNA analysis, for example Northern blot, or a
Southern blot (Sambroolc, J., & Russell, D. W., "Molecular Clofzi.iag", Cold
Spring
Harbor Laboratory Press (2001)), or by using an Invader° RNA Assay
(Third Wave -
www.twt.com). Such methods are based on the hybridisation of a probe (or
primer)
nucleic acid sequence to the target nucleic acid of interest in a sample.
The InvaderR assay is based on a "perfect-match" enzyme-substrate reaction.
Certain
endonuclease enzymes are used which recognise and cut only the specific
structure
formed during the Invader" process. The method relies upon linear
amplification of
the signal generated, rather than on exponential amplification of the target
as in PCR-
based approaches. This allows for easy quantification of the target
concentration and
reduces the effects of sample contamination which may result from exponential
target
amplification. The system is applicable to analysis of RNA and DNA samples. In
the
Invader~ process, two short DNA probes hybridise to the target to form the
structure
recognised by the endonuclease enzymes. The enzyme then cuts one of the probes
to
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release a short DNA sequence. Each target can induce the release of several
thousand
such sequence fragments per hour. Each released sequence fragment can bind to
a
fluorescently-labelled probe and form another cleavage structure. When the
endonuclease cuts the labelled probe, the probe emits a detectable fluorescent
signal.
5 Each released DNA sequence fragment can generate thousands of signals per
hour,
yielding millions of detectable signals per target (Heisler, L. M. & Lonergan,
S. C.,
Biozzzol. Eng. ifz press, (2001); Fors et al Plzaf~nacogeizofzzics, 1 219-229
(2000);
Heisler et al Clifzical Hetzzostasis Review, 14 (11) 10-11 (2000); Leider, K.
W.
Advance for Laboratory Managers 70-71 (February 2000); Treble et al Gefze aizd
Medicii2e 4 68-72 (2000); Leider, K. W. Advance for Laboratory Mafzagers 50-52
(November 1999)).
One of the advantages of an Invader" RNA Assay is that it could be carried out
in a
farm location without complicated equipment or sensitive materials and having
no
need for specialist experience once initial training has been provided. For
example, an
InvaderR RNA Assay may be devised based upon the 324bp FAF fragment. This
could rely on the genomic DNA sequence or on the RNA transcript. The
constituents
of the assay can be provided dried down, in a mufti-well format, such as a 96-
well or
384-well plate. In use the currently available InvaderR RNA Assay would
comprise a
probe/Invader° mix (a FAF probe in the present invention), a signal
probe, a signal
buffer, Cleavase VIII enzyme, a "no target" control, and Rnase-free water. The
biological sample (DNA, RNA, amniotic fluid, allantoic fluid, lysed tissue, or
lysed
blood) can then be added to the wells. The plate can then be incubated in a
standard
water bath for a defined period and then scanned in a fluorescence reader. The
means
for detecting the florescence may by a fluorescence resonance energy transfer
(FRET)
assay.
An RNA-based Invaders assay may be particularly advantageous given the anti-
sense
nature of the FAF sequence. An RNA Invader" assay would have an
oligonucleotide
complementary to the transcribed region so would not bind to anything
transcribed of
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6
the other complementary DNA strand.
The probe to be used in the methods of the invention can be designed according
to the
general principles of the assay system used. The length of the probe used will
depend
on whether the assay system is PCR, Northern blot, Southern blot, or an
Invader"
assay. The probe sequence is at least 6 base pairs (bp) in length and can be
any 6bp
sequence from a nucleic acid sequence as shown in Figures 8 to 14 or a
complementary sequence thereto, as appropriate with respect to the assay
system used.
The sequences of Figures 8 to 14 encode a female specific RNA transcript and
can be
referred to as "Female Associated Factor" or FAF. Such sequences are therefore
target sequences.
The probe sequence can be from 6 to lObp, at least l0bp, or at least l5bp,
lObp to
l5bp, l5bp to 20bp, at least 20bp, 20bp to 25bp and so on up to at least
324bp.
Additional nucleotide residues can be included in the probes designed as
required
provided that no disruption to the binding of probe to target is seen.
So for a Northern or a Southern assay method, a full-length cDNA probe can be
used,
or fragments or oligonucleotides based on the full length sequence. For an
Invader~
assay, probe lengths of from 15 base pairs up to a full length cDNA could be
used
based on any one of the sequences shown in Figures 8 to 14.
The nucleic acid may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Suitably, the nucleic acid is an isolated nucleic acid.
Methods of the present invention can determine whether an individual avian
subject is
male by virtue of the absence of female-specific RNA transcript or DNA
sequence, or
whether the subject is female by virtue of the presence of the female-specific
RNA
transcript or DNA sequence in the sample, in which the female-specific RNA
transcript, or DNA sequence is derived from a sequence of Figures 8 to 14, or
a
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7
sequence complementary or homologous thereto.
The percent identity of two nucleic acid sequences is determined by aligning
the
sequences for optimal comparison purposes (e.g., gaps can be introduced in the
first
sequence for best alignment with the sequence) and comparing the amino acid
residues
or nucleotides at corresponding positions. The "best alignment" is an
alignment of
two sequences which results in he highest percent identity. The percent
identity is
determined by the number of identical amino acid residues or nucleotides in
the
sequences being compared (i.e., % identity = # of identical positions/total #
of
positions x 100).
The determination of percent identity between two sequences can be
accomplished
using a mathematical algorithm known to those of skill in the art. An example
of a
mathematical algorithm for comparing two sequences is the algorithm of Karlin
and
Altschul Pr-~c. Natl. Aead. Sci. USA (1990) 87:2264-2268, modified as in
Karlin and
Altschul (1993) Pf~oc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and
XBLAST programs of Altschul et al, J. Mol. Biol. (1990) 215:403-410 have
incorporated such an algorithm. BLAST nucleotide searches can be performed
with
the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide
sequences
homologous to a nucleic acid molecules of the invention. To obtain gapped
alignments
for comparison purposes, Gapped BLAST can be utilised as described in Altschul
et
al, Nucleic Acids Res. (1997) 25:3389-3402. Alternatively, PSI-Blast can be
used to
perform an iterated search which detects distant relationships between
molecules (Id.).
When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., NBLAST) can be used. See
www.ncbi.nlm.nih.~ov.
Another example of a mathematical algorithm utilised for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN
program (version 2.0) which is part of the GCG sequence alignment software
package
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has incorporated such an algorithm. Other algorithms for sequence analysis
known in
the art include ADVANCE and ADAM as described in Torellis and Robotti Conaput.
Appl. Biosci. (1994) 10:3-5; and FASTA described in Pearson and Lipman Proc.
Natl.
Acad. Sci. USA (1988) 85:2444-8. Within FASTA, ktup is a control option that
sets the
sensitivity and speed of the search.
A nucleic acid sequence which is complementary to a nucleic acid sequence
useful in
a method of the present invention is a sequence which hybridises to such a
sequence
under stringent conditions, or a nucleic acid sequence which is homologous to
or
would hybridise under stringent conditions to such a sequence but for the
degeneracy
of the genetic code, or an oligonucleotide sequence specific for any such
sequence.
The nucleic acid sequences include oligonucleotides composed of nucleotides
and also
those composed of peptide nucleic acids. Where the nucleic sequence is based
on a
fragment of the sequences of the invention, the fragment may be at least any
ten
consecutive nucleotides from the gene, or for example an oligonucleotide
composed of
from 20; 30, 40, or 50 nucleotides.
Stringent conditions of hybridisation may be characterised by low salt
concentrations
or high temperature conditions. For example, highly stringent conditions can
be
defined as being hybridisation to DNA bound to a solid support in 0.5M NaHP04,
7%
sodium dodecyl sulfate (SDS), 1mM EDTA at 65°C, and washing in O.IxSSC/
0.1 %SDS at 68°C (Ausubel et al eds. "Cur-reiat Protocols i~z Molecular
Biology" 1,
page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley &
Sons,
Inc., New York, (1989)). In some circumstances less stringent conditions may
be
required. As used in the present application, moderately stringent conditions
can be
defined as comprising washing in 0.2xSSC/0.1%SDS at 42°C (Ausubel et al
(1989)
supra). Hybridisation can also be made more stringent by the addition of
increasing
amounts of formamide to destabilise the hybrid nucleic acid duplex. Thus
particular
hybridisation conditions can readily be manipulated, and will generally be
selected
according to the desired results. In general, convenient hybridisation
temperatures in
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9
the presence of 50% formamide are 42°C for a probe which is 95 to 100%
homologous
to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70
to 90% homology.
Examples of preferred nucleic acid sequences for use in a method of the
present
invention are the sequences of the invention shown in Figures 8 to 14.
Complementary or homologous sequences may be 75%, 80%, 85%, 90%, 95%, 99%
similar to such sequences.
The advantages of the methods of the present invention are that the sex of an
avian
subject can be readily and easily determined based on a single biological
sample.
There are immediate animal welfare implications in agriculture as the previous
practice of whole chick homogenisation can be discontinued.
In a preferred embodiment of the invention there is provided a method for
determining
the sex of an avian subject, the method comprising the steps of:
(1) obtaining a suitable sample from an avian subject being
(i) an avian embryo iiz ovo; or
(ii) an individual avian
(2) preparing sample for analysis;
(3) probing the sample with a nucleic acid probe based on a sequence of
Figures 8 to 14; and
(4) analysing the results of step (3) to determine if individual is male or
female.
In a further preferred embodiment of the invention, there is provided a method
for
determining the sex of an avian embryo, the method comprising the steps of:
(1) obtaining a suitable sample from an avian egg
(2) preparing sample for analysis;
(3) probing the sample with a nucleic acid probe based on a sequence of
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Figures 8 to 14; and
(4) analysing the results of step (3) to determine if individual is male or
female.
In certain embodiments of the invention, the use of the Invader° assay
may be
5 preferable. It may also be advantageous to analyse RNA transcripts present
in the
sample using the Invaderrt assay. Alternatively, the Polymerase Chain Reaction
(PCR) may be used, either standard PCR and gel analysis, or a quantitative PCR
analysis such as Taqman ~i .
10 According to a second aspect of the invention, there is provided the use of
a nucleic
acid sequence or a fragment thereof according to any one of Figures 8 to 14 in
a
method according to the first aspect of the invention.
According to a third aspect of the invention there is provided a nucleic acid
sequence
as shown in any one of Figures 8 to 14. Such isolated sequences have use in
methods
and uses in accordance with the first and second aspects of the invention in
determining the sex of an avian subject.
According to a fourth aspect of the invention there is provided a kit of parts
comprising a nucleic acid probe comprising an at least 6 base pair fragment
from a
nucleic acid sequence as shown in any one of Figures 8 to 14 for determining
the sex
of an avian subject or a sequence complementary or homologous thereto. The kit
may
further comprise instructions for use according to a method of the invention.
The
probe may suitably be provided in a removably sealed container.
According to a fifth aspect of the invention, there is provided a polypeptide
or
fragment thereof coded for by a nucleic acid sequence of any one of Figures 8
to 12.
The term "polypeptide" includes both peptide and protein, unless the context
specifies
otherwise. Examples of such peptide sequences are shown in Figure 15.
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11
Such polypeptides include analogues, homologues, orthologues, isoforms,
derivatives,
fusion proteins and proteins with a similar structure or are a related
polypeptide as
herein defined.
The temp "analogue" as used herein refers to a polypeptide that possesses a
similar or
identical function as a protein coded for by a nucleic acid sequence of the
invention
but need not necessarily comprise an amino acid sequence that is similar or
identical to
an amino acid sequence of the invention, or possess a structure that is
similar or
identical to that of protein of the invention. As used herein, an amino acid
sequence
of a polypeptide is "similar" to that of a polypeptide of the invention if it
satisfies at
least one of the following criteria: (a) the polypeptide has an amino acid
sequence that
is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at
least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at
least 85%, at least 90%, at least 95% or at least 99%) identical to the amino
acid
sequence of a polypeptide of the present invention; (b) the polypeptide is
encoded by a
nucleotide sequence that hybridizes under stringent conditions to a nucleotide
sequence encoding at least 5 amino acid residues (more preferably, at least 10
amino
acid residues, at least 15 amino acid residues, at least 20 amino acid
residues, at least
amino acid residues, at least 40 amino acid residues, at least 50 amino acid
20 residues, at least 60 amino residues, at least 70 amino acid residues, at
least 80 amino
acid residues, at least 90 amino acid residues, at least 100 amino acid
residues, at least
125 amino acid residues, or at least 150 amino acid residues) of a polypeptide
sequence of the invention; or (c) the polypeptide is encoded by a nucleotide
sequence
that is at least 30% (more preferably, at least 35%, at least 40%, at least
45%, at least
25 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%,
at least 85%, at least 90%, at least 95% or at least 99%) identical to the
nucleotide
sequence encoding a polypeptide of the invention.
As used herein, a polypeptide with "similar structure" to that of a
polypeptide of the
invention refers to a polypeptide that has a similar secondary, tertiary or
quaternary
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12
structure as that of a polypeptide of the invention. The structure of a
polypeptide can
determined by methods known to those skilled in the art, including but not
limited to,
X-ray crystallography, nuclear magnetic resonance, and crystallographic
electron
microscopy.
The term "fusion protein" as used herein refers to a polypeptide that
comprises (i) an
amino acid sequence of a polypeptide of the invention, a fragment thereof, a
related
polypeptide or a fragment thereof and (ii) an amino acid sequence of a
heterologous
polypeptide (i.e., not a polypeptide sequence of the present invention).
The term "homologue" as used herein refers to a polypeptide that comprises an
amino
acid sequence similar to that of a protein of the invention but does not
necessarily
possess a similar or identical function.
The term "orthologue" as used herein refers to a non-human polypeptide that
(i)
comprises an amino acid sequence similar to that of a protein of the invention
and (ii)
possesses a similar or identical function.
The term "related polypeptide" as used herein refers to a homologue, an
analogue, an
isoform of , an orthologue, or any combination thereof of a protein of the
invention.
The term "derivative" as used herein refers to a polypeptide that comprises an
amino
acid sequence of a polypeptide of the invention which has been altered by the
introduction of amino acid residue substitutions, deletions or additions. The
derivative
polypeptide possess a similar or identical function as polypeptides of the
invention.
The term "fragment" as used herein refers to a peptide or polypeptide
comprising an
amino acid sequence of at least 5 amino acid residues (preferably. at least 10
amino
acid residues, at least 15 amino acid residues, at least 20 amino acid
residues, at least
25 amino acid residues, at least 40 amino acid residues, at least 50 amino
acid
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13
residues, at least 60 amino residues, at least 70 amino acid residues, at
least 80 amino
acid residues, at least 90 amino acid residues, at least 100 amino acid
residues) of the
amino acid sequence of a polypeptide of the invention. The fragment of may or
may
not possess a functional activity of such polypeptides.
The term "isoform" as used herein refers to variants of a polypeptide that are
encoded
by the same gene, but that differ in their isoelectric point (pI) or molecular
weight
(MW), or both. Such isoforms can differ in their amino acid composition (e.g.
as a
result of alternative splicing or limited proteolysis) and in addition, or in
the
alternative, may arise from differential post-translational modification
(e.g.,
glycosylation, acylation, phosphorylation). As used herein, the term "isoform"
also
refers to a protein that exists in only a single form, i.e., it is not
expressed as several
variants.
The percent identity of two amino acid sequences or of two nucleic acid
sequences is
determined by aligning the sequences for optimal comparison purposes (e.g.,
gaps can
be introduced in the first sequence for best alignment with the sequence) and
comparing the amino acid residues or nucleotides at corresponding positions.
The
"best alignment" is an alignment of two sequences which results in the highest
percent
identity. The percent identity is determined by the number of identical amino
acid
residues or nucleotides in the sequences being compared (i.e., °Io
identity = # of
identical positions/total # of positions x 100).
The determination of percent identity between two sequences can be
accomplished
using a mathematical algorithm known to those of skill in the art. An example
of a
mathematical algorithm for comparing two sequences is the algorithm of Marlin
and
Altschul Pr-oc. Natl. Acad. Sci. USA (1990) 87:2264-2268, modified as in
Marlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and
XBLAST programs of Altschul et al, J. Mol. Bi~l. (1990) 215:403-410 have
incorporated such an algorithm. BLAST nucleotide searches can be performed
with
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14
the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide
sequences
homologous to a nucleic acid molecules of the invention. BLAST protein
searches
can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain
amino acid sequences homologous to a protein molecules of the invention. To
obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilised as
described in Altschul et al, Nucleic Aciels Res. (1997) 25:3389-3402.
Alternatively,
PSI-Blast can be used to perform an iterated search which detects distant
relationships
between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another example of a mathematical algorithm utilised for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN
program (version 2.0) which is part of the GCG sequence alignment software
package
has incorporated such an algorithm. Other algorithms for sequence analysis
known in
the art include ADVANCE and ADAM as described in Torellis and Robotti
Cof~iput.
Appl.. Biosci. (1994) 10:3-5; and FASTA described in Pearson and Lipman Proc.
Natl.
Acad. Sci. USA (1988) 85:2444-8. Within FASTA, ktup is a control option that
sets the
sensitivity and speed of the search.
The skilled person is aware that various amino acids have similar properties.
One or more
such amino acids of a substance can often be substituted by one or more other
such
amino acids without eliminating a desired activity of that substance. Thus the
amino
acids glycine, alanine, valine, leucine and isoleucine can often be
substituted for one
another (amino acids having aliphatic side chains). Of these possible
substitutions it is
preferred that glycine and alanine are used to substitute for one another
(since they have
relatively short side chains) and that valine, leucine and isoleucine are used
to substitute
for one another (since they have larger aliphatic side chains which are
hydrophobic).
Other amino acids which can often be substituted for one another include:
phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains); lysine,
arginine and
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histidine (amino acids having basic side chains); aspartate and glutamate
(amino acids
having acidic side chains); asparagine and glutamine (amino acids having amide
side
chains); and cysteine and methionine (amino acids having sulphur containing
side
chains). Substitutions of this nature are often referred to as "conservative"
or "semi-
5 conservative" amino acid substitutions.
Amino acid deletions or insertions may also be made relative to the amino acid
sequence
of a polypeptide sequence of the invention. Thus, for example, amino acids
which do
not have a substantial effect on the activity of such polypeptides, or at
least which do not
10 eliminate such activity, may be deleted. Amino acid insertions relative to
the sequence of
polypeptides of the invention can also be made . This may be done to alter the
properties
of a protein of the present invention (e.g. to assist in identification,
purification or
expression, where the protein is obtained from a recombinant source, including
a fusion
protein. Such amino acid changes relative to the sequence of a polypeptide of
the
15 invention from a recombinant source can be made using any suitable
technique e.g. by
using site-directed mutagenesis. The molecule may, of course, be prepared by
standard
chemical synthetic techniques, e.g. solid phase peptide synthesis, or by
available
biochemical techniques.
It should be appreciated that amino acid substitutions or insertions within
the scope of the
present invention can be made using naturally occurring or non-naturally
occurring
amino acids. Whether or not natural or synthetic amino acids are used, it is
preferred that
only L-amino acids are present.
According to a sixth aspect of the invention, there is provided a vector
comprising a
nucleic acid sequence of any one Figures 8 to 12. The term "vector" or
"expression
vector" generally refers to any nucleic acid vector which may be RNA, DNA or
cDNA.
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16
The term "expression vector" may include, among others, chromosomal, episomal,
and virus-derived vectors, for example, vectors derived from bacterial
plasmids, from
bacteriophage, from transposons, from yeast episomes, from insertion elements,
from
yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such
as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and
retroviruses, and vectors derived from combinations thereof, such as those
derived
from plasmid and bacteriophage genetic elements, such as cosmids and
phagemids.
Generally, any vector suitable to maintain, propogate or express nucleic acid
to
express a polypeptide in a host may be used for expression in this regard.
In certain embodiments of the invention, the vectors may provide for specific
expression. Such specific expression may be inducible expression or expression
only
in certain types of cells or both inducible and cell-specific. Preferred among
inducible
vectors are vectors that can be induced for expression by environmental
factors that
are easy to manipulate, such as temperature and nutrient additives.
Particularly
preferred among inducible vectors are vectors that can be induced for
expression by
changes in the levels of chemicals, for example, chemical additives such as
antibiotics.
A variety of vectors suitable for use in the invention, including constitutive
and
inducible expression vectors for use in prolearyotic and eukaryotic hosts, are
well
known and employed routinely by those skilled in the art.
Recombinant expression vectors will include, for example, origins of
replication, a
promoter preferably derived from a highly expressed gene to direct
transcription of a
structural sequence, and a selectable marker to permit isolation of vector
containing
cells after exposure to the vector.
Expression vectors may comprise an origin of replication, a suitable promoter
and
enhancer, and also any necessary ribosome binding sites, polyadenylation
regions,
splice donor and acceptor sites, transcriptional termination sequences, and 5'-
flanking
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17
non-transcribed sequences that are necessary for expression. Preferred
expression
vectors according to the present invention may be devoid of enhancer elements.
The promoter sequence may be any suitable known promoter, for example the
human
cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV
thymidine kinase promoter, the early and late SV40 promoters or the promoters
of
retroviral LTR's, such as those of the Rous sarcoma virus ("RSV"), and
metallothionein promoters, such as the mouse metallothionein-I promoter. The
promoter may comprise the minimum sequence required for promoter activity
(such as
a TATA box without enhancer elements), for example, the minimal sequence of
the
CMV promoter (mCMV).
The expression vectors may also include selectable markers, such as antibiotic
resistance, which enable the vectors to be propagated.
The nucleic acid sequence contained in the expression vector of this aspect of
the
invention may be a reporter transcription unit lacking a promoter region, such
as a
chloramphenicol acetyl transferase ("CAT") transcription unit. As is well
known,
introduction into an. expression vector of a promoter-containing fragment at a
restriction site upstream of the CAT gene engenders the production of CAT
activity,
which can be detected by standard CAT assays. The application of reporter
genes
relates to the phenotype of these genes which can be assayed in a transformed
organism and which is used, for example, to analyse the induction and/or
repression of
gene expression. Reporter genes for use in studies of gene regulation include
other
well known reporter genes including the lux gene encoding luciferase which can
be
assayed by a bioluminescence assay, the uidA gene encoding (3-glucuronidase
which
can be assayed by a histochemical test, the aphlV gene encoding hygromycin
phosphotransferase which can be assayed by testing for hygromycin resistance
in the
transformed organism, the dhfi- gene encoding dihydrofolate reductase which
can be
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18
assayed by testing for methotrexate resistance in the transformed organism,
the ~aeo
gene encoding neomycin phosphotransferase which can be assayed by testing for
kanamycin resistance in the transformed organism and the lacZ gene encoding ~i-
galactosidase which can be assayed by a histochemical test. All of these
reporter
genes are obtainable from E.coli except for the lux gene. Sources of the lux
gene
include the luminescent bacteria Vibj~io harweyii and V.fischeri, the firefly
Plzotiraus
pyralis and the marine organism Re~ailla rerzifonrais.
According to a seventh aspect of the invention, there is provided a host cell
comprising a vector as described above. Suitably, the host cell is stably
transfected by
the vector. The nucleic acid sequence of the invention may be incorporated
into the
genome of the cell or it may be expressed episomally. In general, the host
cell will be
of an avian species as described herein, but other host cells such as bacteria
or yeast
are included within the scope of this aspect of the invention.
Introduction of an expression vector into the host cell can be effected by
calcium
phosphate transfection, DEAF-dextran mediated transfection, microinjection,
cationic
lipid-mediated transfection, electroporation, transduction, scrape loading,
ballistic
introduction, infection of other methods. Such methods are described in many
standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2°d Ed., Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, N.Y. (1989).
According to an eighth aspect of the invention, there is provided an antibody
to a
polypeptide of the fifth aspect of the invention.
The antibody may be a polyclonal antibody or a monoclonal antibody. Polyclonal
antibodies can be raised by stimulating their production in a suitable animal
host (e.g. a
mouse, rat, guinea pig, rabbit, sheep, chicken, goat or monkey) when the
substance of the
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19
present invention is injected into the animal. If necessary an adjuvant may be
administered together with the substance of the present invention. The
antibodies can
then be purified by virtue of their binding to a protein of the invention or
as described
further below. Monoclonal antibodies can be produced from hybridomas. These
can be
formed by fusing myeloma cells and spleen cells which produce the desired
antibody in
order to form an immortal cell line. This is the well known Kohler & Milstein
technique
(Nature 256 52-55 (1975)).
Techniques for producing monoclonal and polyclonal antibodies which bind to a
particular protein are now well developed in the art. They are discussed in
standard
immunology textbooks, for example in Roitt et al, Inzzzzuzzology second
edition (1989),
Churchill Livingstone, London.
In addition to whole antibodies, the present invention includes derivatives
thereof which
are capable of binding to a polypeptide of the invention. Thus the present
invention
includes antibody fragments and synthetic constructs. Examples of antibody
fragments
and synthetic constructs are given by Dougall et al in Tibteclz 12 372-379
(September
1994). Antibody fragments include, for example, Fab, F(ab')2 and Fv fragments
(see
Roitt et al [supra]). Fv fragments can be modified to produce a synthetic
construct
known as a single chain Fv (scFv) molecule. This includes a peptide linker
covalently
joining Vh and V~ regions which contribute to the stability of the molecule.
Other synthetic constructs include CDR peptides. These are synthetic peptides
comprising antigen binding determinants. Peptide mimetics may also be used.
These
molecules are usually conformationally restricted organic rings which mimic
the
structure of a CDR loop and which include antigen-interactive side chains.
Synthetic
constructs also include chimaeric molecules. Thus, for example, humanised (or
primatised) antibodies or derivatives thereof are within the scope of the
present invention.
An example of a humanised antibody is an antibody having human framework
regions,
but rodent hypervariable regions. Synthetic constructs also include molecules
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comprising a covalently linked moiety which provides the molecule with some
desirable
property in addition to antigen binding. For example the moiety may be a label
(e.g. a
detectable label, such as a fluorescent or radioactive label) or a
pharmaceutically active
agent.
5
The antibodies or derivatives thereof specific for a polypeptide of the
invention have a
variety of other uses. They can be used in purification and/or identification
of such
proteins or a cell that expresses the polypeptides. As a result they may be
used in a
diagnostic method according to the present invention.
After the preparation of a suitable antibody to a protein of the invention, it
may be
isolated or purified by one of several techniques commonly available (for
example, as
described in Antibodies: A Laborat~ry Ma~zual, Harlow and Lane, eds. Cold
Spring
Harbor Laboratory Press (1988)). Generally suitable techniques include peptide
or
protein affinity columns, HPLC or RP-HPLC, purification on Protein A or
Protein G
columns, or combinations of these techniques. Recombinant antibodies to
polypeptides of the invention can be prepared according to standard methods,
and
assayed for specificity for these proteins using procedures generally
available,
including ELISA, ABC, dot-blot assays etc.
According to a ninth aspect of the invention, there is provided a method for
the
determination of the sex of an avian subject, the method comprising contacting
a
sample from said subject with an antibody to a polypeptide of the fifth aspect
of the
invention. Suitably the antibody is detestably labelled or is itself contacted
by a
reporter antibody that is detestably labelled. For example the label may be a
fluorescent
or radioactive label. Alternatively, the antibody may be detected by an anti-
idiotype
antibody which is labelled.
According to a tenth aspect of the invention, there is provided a kit of parts
comprising
an antibody as defined above for determining the sex of an avian subject.
Suitably, the
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21
antibody is supplied in a removably sealed container. The kit may further
comprise
instructions for use according to a method of the invention.
Preferred features for the second and subsequent aspects of the invention are
as for the
first aspect nautatis nautatadis.
The invention will now be further described by way of reference to the
following
Examples and Figures which are provided for the purposes of illustration only
and are
not to be construed as being limiting on the invention. Reference is made to a
number
of Figures in which:
FIGURE 1 shows the results of Northern analysis of RNA samples from a day
4.5 whole chick embryos using differential display clone 378.2.6 as a probe.
Two major bands were detected in females and transcript sizes were
approximately 800bp and 1300bp.
FIGURE 2 shows the results of a Southern blot of male and female chicken
genomic DNA digested with four different restriction enzymes probed with
3zP-labelled cDNA clone 378.2.6
FIGURE 3 shows the results of W-specific PCR at 57°C, 55°C
and 53°C using
PCR primers designed from the 796bp sequence of FAF 4.
FIGURE 4 shows the results of a northern blot of total RNA from male and
female tissues - heart, brain, lung, liver, chorioallantoic membrane and
mesonephrous- probed with 32P-labelled clone 378.2.6.
FIGURE 5(a) shows the position of the FAF-4 796bp sequence in relation to
the w pkci gene. FIGURE 5(b) shows the relative position of the PCR 204bp
product with respect to the FAF-4 796bp sequence clone.
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FIGURE 6 shows a species blot probed with FAF display fragment for
chiclcen, quail and turkey.
FIGURE 7 shows a diagrammatical representation of sampling of amnion and
allantois of a developing chick embryo.
FIGURE 8 shows the nucleotide sequence for the differential display fragment
of 324bp (FAF-1).
FIGURE 9 shows the nucleotide sequence of FAF-2 of 796bp.
FIGURE 10 shows the nucleotide sequence of FAF-3 of 772bp.
FIGURE 11 shows the nucleotide sequence of FAF-4 of 796bp.
FIGURE 12 shows the nucleotide sequence of FAF-5 of 1283bp.
FIGURE 13 shows a fragment of the nucleotide sequence of FAF from Turkey.
FIGURE 14 shows a fragment of the nucleotide sequence of FAF from Quail.
FIGURE 15 shows the putative ORFs for isolated chicken FAF clones: Figure
15(a) shows the putative ORFs for FAFl, Figure 15(b) for FAF2, Figure 15(c)
for FAF3, Figure 15(d) for FAF4 and Figure 15(e) for FAFS.
Example l: Differential Display Reverse Transcriptase - Polymerase Chain
Reaction
(DDRT-PCR)
DDRT-PCR is a powerful molecular tool which allows visualisation of gene
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23
expression in any particular cell type or tissue via the creation of RNA
fingerprints.
Genes which are differentially expressed between two or more samples under
study
are readily identifiable and recoverable using this technique. Bands
representing
differentially expressed cDNAs (for example, in male and female tissues) can
be
recovered and cloned (Miele et al In "Expressioa2 Ge~zetics", eds. McClelland
&
Pardee, pages 433-444, Natick: Eaton Publishing (1999)(a); Miele et al Prep.
Bioclaena. Biotech. 29(3) 245-255 (1990)(b)). Cloned cDNAs are sequenced and
identified following computer-assisted homology searching of the public
nucleotide
and protein databases. Cloned cDNAs were used for radiolabelling for use as
probes
in Northern and Southern hybridisation studies according to standard
protocols.
Differential display analysis of RNA from male and female whole chicken
embryos
harvested on days 2.5, 3, 3.5, 4, and 4.5 using primers dTl2-MC (M=A,G,C) and
DM8
(AGTGCCGTTA) revealed two bands which appeared to be female specific. These
bands were cut out from the display gel, re-amplified using primers containing
EcoRl
restriction sites and cloned into EcoR1 digested pBILSK+. Colonies obtained
were
screened for inserts, by PCR, using T7 and T3 primers. Two positive clones,
378.2.2
and 378.2.6 were obtained having insert sizes of approximately 350bp, roughly
the
size of insert expected from the bands cut from the display gel. A fraction of
the
display reactions were run on an agarose/TBE gel and Southern blotted (Miele
et al
2000). 32P-labelled inserts from the isolated differential display clones were
used to
probe the blots. They gave the same female specific banding pattern,
confirming that
they corresponded to the cDNA bands cut from the display gel. Sequence
analysis of
the two clones revealed that they were identical.
Northern hybridisation is used to measure the amount and size of RNAs
transcribed
from eukaryotic genes. After isolating intact mRNA sequences, representing the
products of Gene transcription, the fragments can be separated and immobilised
in a
similar way to DNA sequences in Southern hybridisation. Major differences
include
the need for scrupulous handling to avoid degradation of the RNA by enzymes
and the
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use of denaturing agents such as formamide to preserve the single-stranded,
linear
nature of the transcripts and allow them to be separated on the basis of their
size.
(Sambrook, J. & Russell, D. W.,"Molecular Clofzi~zg: a Laboratofy Mafzual" 3'd
edition, New Yorlc: Cold Spring Harbor Laboratory Press (2001)).
RNA samples from pooled male and pooled female whole chick embryos, days 2.5,
3,
3.5 and 4.5 were used to prepare northern blots. When differential display
clone
378.2.2 was used as a probe, two major bands were detected in females.
Transcript
sizes were approximately 800bp, 1300bp. No bands were apparent in the male
samples. The results are shown in Figure 1.
Example 2: Southern Hybridisation
Southern transfer is used to study how genes are organised within genomes
using
specific probes that hybridise to a portion of the gene. The genomic DNA is
digested
with restriction enzymes which cut at specific sites and produce a range of
fragments
of different sizes. The digested DNA is added to wells at one end of an
agarose gel.
Under the influence of an electric potential the DNA moves down the gel in
columns,
the fragments becoming separated by size, the smaller fragments moving more
quickly. The DNA is transferred from the gel by blotting onto a solid support,
such as
a nylon membrane. This is then labelled with the radioactive probe which
hybridises
to the complementary sequences. These can be visualised as dark bands on a
photographic negative in the process of autoradiography. (Sambrook, J. &
Russell, D.
W., "Molecular Cloni~zg: a Laboratory Manual" 3rd edition, New York: Cold
Spring
Harbor Laboratory Press (2001)).
A Southern blot of male and female chicken genomic DNA, digested with four
different restriction enzymes, was probed with the 32P labelled cDNA clone
378.2.6.
A positive signal was seen in the female samples but no bands were detected in
male
samples even after the blots were overexposed. The results are shown in Figure
2.
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Example 3: W-specific PCR
PCR primers were designed from the 796 by sequence of FAF-4. The sequences of
5 the primers were FAF-Forward primer 5'-AGAATAAACGCCCCTCGATT-3', and
FAF reverse primer, 5'-CAGGTTCTCTTTCTCGGTCG-3'. PCR reactions were
performed in 25,1 lOmM Tris-HCI, l.5mM MgCl2, 50mM I~C1 pH8.3 containing
200~M dNTP's, 0.8~.M primers and lU Taq polymerase. Following an initial
denaturation step of 2 minutes at 94°C, DNA was denatured at
94°C for 30 seconds,
10 annealed at 50°C, or 53°C, or 57°C for 30 seconds and
extended at 72°C for 30
seconds. Reactions were subjected to 30 cycles of amplification. A final
extension
step at 72°C for 5 minutes was carried out. After amplification, 20,1
of reaction mix
was loaded onto a 1 % TBE / agarose gel and electrophoresed for 1 hour at 100
volts.
The results are shown in Figure 3.
The PCR reaction described in this Example amplifies part of the conserved
[324bp]-
nucleotide sequence of FAF which is present on the W-chromosome but not the Z-
chromosome. Figure 3 shows that for the three annealing temperatures in the
region
of the melting temperature of these two primers, there is amplification of the
sequence
in the female but not male samples. This specificity is maintained even at
lower
temperatures which increase the possibility of non-specific binding. The
results
demonstrate that the PCR method can successfully be applied to distinguish
unambiguously between male and female DNA.
Example 4: Analysis of expression in day 11 chick tissues
A northern blot of total RNA from male and female muscle, heart, brain, lung,
liver,
chc~ri~allantoic membrane and mesonephrous was probed with 3zP-labelled clone
378.2.6. The female specific banding patterns obtained were identical in all
tissues
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26
tested, differing only in the level of expression. The results are shown in
Figure 4.
Example 5: Analysis of location of FAF-4 796 by fragment
Figure 5(a) shows the position of the [FAF]8 796bp sequence in relation to the
w-plcci
gene and Figure 5(b) shows the relative position of the PCR 204bp product with
respect to the [FAF]8 796bp sequence clone. The forward primer (A) is
5'-AGAATAAACGCCCCTCGATT-3'
The reverse primer (B) is
5'-CAGGTTCTCTTTCTCGGTCG-3'
Primer details:
Oligo Start Length Tm GC% Any 3'
Left primer414 20 59.93 45.00 4.00 2.00
Right primer617 20 59.98 55.00 2.00 2.00
Example 6: Species blot
The results of a species blot probed with FAF display fragment are shown in
Figure 6.
The samples probed were obtained from chicken, quail and turkey. Standard
genomic
DNA extraction from blood from these three species was followed by standard
Southern analysis using the original 324bp FAF-1 fragment as a probe.
Example 7' Sam~lin~ of amnion and allantois of a developing chick embryo
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In order to perform a method of the invention for the purposes of determining
the sex
of a chick embryo, it is necessary to obtain samples of the amnion and/or
allantois of
the embryo inside the egg. Small volumes (5~.1 to 25,1) of amniotic and
allantoic fluid
can be removed manually or by automated sampling. The collected fluids can
then be
used as substrates in chick-sexing PCR methods according to the present
invention.
Sampling of fluids from the chiclc embryo is shown diagrammatically in Figure
7.
Example 8: Sequencing of differentiall~pressed RNAs
The sequence of the insert of approximately 350bp found in the two positive
clones,
378.2.2 and 378.2.6 identified in Example 1 was sequenced by the method of
dideoxy
chain termination analysis. The sequence of the FAF-display fragment is shown
in
Figure 8.
The DNA sequences corresponding to the approximately 800bp and 1300bp RNA
bands identified in Example 1 were sequenced as above. The results are shown
in
Figures 9 to 12 as sequences FAF-2, FAF-3, FAF-4 and FAF-5.
The sequence information obtained was subjected to further analysis to look
for
homology with other sequences in the available databases and to compare the
different
sequences with each other.
The differential display fragment of 350bp was found not to match exactly with
any of
FAF-2, FAF-3, FAF-4, or FAF-5, but a considerable degree of overlap was seen
with
only relatively few base pair substitutions, or gaps.
In the comparison of the other sequences, FAF-2 was found to show only a 2
nucleotide difference from FAF-5 and FAF-4 when the sequences were aligned.
Sequence FAF-2 has a 90rn~ homology with sequence FAF-3 and sequence FAF-4
matches FAF-5 exactly over 796 nucleotides.
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In a BLAST 2.2.1 search on www.ncbi.nlm.nih.govv, it was found that FAF-4 has
four
nucleotide differences with the 5' genomic non-translated region of the ulpcki
gene.
The search as a BLAST search of the nr (non-redundant) nucleotide databases.
It is concluded that FAF is located on the complementary strand of the wpkci
repeat
region, in which there are approximately 40 repeats of the wpcki gene.
However, it
lies in the inter-genic region of those repeats with less well conserved
sequences. Four
FAF transcripts have now been identified and slight differences in the
sequence (or in
the case of FAF-4 and FAF-5, the different lengths) suggests that they each
come from
a different repeat of the gene.
Example 9: Predicted protein coding sequences and antibodies
Analysis of the FAF sequences shows that open-reading frames encoding proteins
exist. The FAF peptides of the ORFs are shown in Figure 15.
The FAF sequences are expressed in a suitable host cell, typically an avian in
vitro
culture system such as the method of Perry et al (EP-A-0295964) and purified
using
affinity chromatography. Purified FAF peptides, fragments or fusion proteins
thereof
can be used to generate monoclonal antibodies against FAF using conventional
techniques, for example those described in A~atibodies: A Laboratory Mayzual,
Harlow
and Lane, eds. Cold Spring Harbor Laboratory Press (1988)).
Briefly, mice are immunised with a FAF peptide as an immunogen emulsified in
complete Freund's adjuvant, and injected in amounts ranging from 10-100~g
subcutaneously or intraperitoneally. Ten to twelve days later, the immunised
animals
are boosted with additional FAF peptide emulsified in incomplete Freund's
adjuvant.
Mice are periodically boosted thereafter on a weekly to bi-weekly immunisation
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29
schedule. Serum samples are periodically taken by retro-orbital bleeding or
tail-tip
excision to test for anti-FAF peptide antibodies by dot blot assay, or ELISA.
Following detection of an appropriate antibody titre, positive animals are
provided one
last intravenous injection of FAF peptide in saline. Three to four days later,
the
animals are sacrificed , spleen cells harvested, and spleen cells are fused to
a murine
myeloma cell line, e.g. NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580).
Fusions
generate hybridoma cells, which are plated in multiple microtitre plates in a
HAT
(hypoxanthine, aminopterin and thymidine) selective medium to inhibit
proliferation
of non-fused cells, myeloma hybrids and spleen cell hybrids.
The hybridoma cells are screened by ELISA for reactivity against purified FAF
peptides by adaptations of the techniques described in Engvall et al
(Inz~zzufzoclzef~z. 8,
871 (1971)) or Beckmann et al (J. Ioz~zzmzol. 144, 4212 (1990)). Positive
hybridoma
cells can be injected into syngeneic BALB/c mice to produce ascites containing
high
concentrations of anti-FAF monoclonal antibodies. Alternatively, hybridoma
cells can
be grown irz vitro in flasks or roller bottles by various techniques.
Monoclonal
antibodies produced in mouse ascites can be purified by ammonium sulphate
precipitation, followed by gel exclusion chromatography. Alternatively,
affinity
chromatography based upon binding of antibody to protein A or protein G can
also be
used, as can affinity chromatography based upon binding to FAF peptides. The
resultant antibodies may be suitably stored in a physiological solution, such
as
phosphate buffered saline.
